Abstract

In the past, aligning the counterpropagating beams in our 3D real-time generalized phase contrast (GPC) trapping system has been a task requiring moderate skills and prior experience with optical instrumentation. A ray transfer matrix analysis and computer-controlled actuation of mirrors, objective, and sample stage has made this process user friendly. The alignment procedure can now be done in a very short time with just a few drag-and-drop tasks in the user-interface. The future inclusion of an image recognition algorithm will allow the alignment process to be executed completely without any user interaction. An automated sample loading tray with a loading precision of a few microns has also been added to simplify the switching of samples under study. These enhancements have significantly reduced the level of skill and experience required to operate the system, thus making the GPC-based micromanipulation system more accessible to people with little or no technical expertise in optics.

Misalignment of a pair of counterpropagating beams results in a trap without a stable fix point as sketched above. An object is first trapped in one beam, pushed out of focus, trapped by the other beam, and pushed back in a looping motion. Arrows indicate the propagation directions of beams.

The setup described in detail in our previous paper [8] has been modified with four motorized mirror mounts (mirrors marked M and DM). Tilting a mirror an angle θ/2 tilts the reflected beam an angle θ. Furthermore, the lower objective has been mounted on a computer-controlled stage, and the sample stage is also computer-controlled on an x, y, z stage. Lenses marked L1 are achromatic with focal lengths (f1) 400 mm. Objectives marked L2 are long working distance objectives with 50x magnification (NA = 0.55, focal lengths (f2) 3.6 mm).

In the ideal geometry for beam alignment, kinematic mirror mounts should be positioned at the “object” plane and at the Fourier plane in a 4-f setup. Tilting a mirror placed in the “object” plane will introduce tilt at the image plane. Adjusting a mirror placed in the Fourier plane results in a translation at the image plane.

Tilting “Mirror M” alone produces the blue beam path (both tilt and displacement in the image plane at the sample), but if “Mirror DM” is accordingly adjusted one can obtain either the green beam path, where the beam hits the original target with an altered tilt, or “Mirror DM” can be adjusted resulting in the beam following the red path, where no beam tilt is induced, but only a displacement of it. All angles and displacements are exaggerated for clarity.

The user is guided through the calibration scheme sketched above. The user is asked to click a “next” button when each step is completed to his or her satisfaction. The computer readies for the next step in the alignment procedure. The text in blue indicates actions presently requiring user attention, whereas the black text (here axial displacement of sample) is automatically taken care of, when the user clicks “next” in the graphical user interface (GUI).

Once the top beam has been aligned; the computer turns off the top beam and turns on the beam coming from below, and guides the user in going through the steps sketched above. The text in blue indicates actions presently requiring user attention, whereas the black text (here defocusing of objective) is automatically taken care of, as the user clicks “next” in the graphical user interface (GUI).

(AVI: 2.3 MB) Movie of beam alignment. The frames above show the focused and defocused crosshairs before and after alignment. In the video we show the alignment being performed, and that the crosshair is indeed aligned. The video is real time, and the alignment of the top beam is completed in less than 20 seconds. [Media 1]

CD-ROM drive customized to work as sample loading system. The large working distance between objective lenses and the absence of high-NA immersion liquid, allow for the insertion of an automated sample loading device as demonstrated.